EstA protein, a novel virulence factor of Streptococcus pneumoniae, induces nitric oxide and pro-inflammatory cytokine production in RAW 264.7 macrophages through NF-kappaB/MAPK.

In the present study we characterized the molecular mechanism by which esterase A (EstA) protein, a novel virulence factor of Streptococcus pneumoniae induces inflammation. Stimulation of RAW 264.7 macrophages with purified EstA protein induced the expression of inducible nitrogen oxide synthase (iNOS) mRNA and nitrogen oxide (NO) production in a concentration-dependent manner. Inhibitors of iNOS, NF-kappaB, p38 and ERK 1/2 MAPK pathways significantly decreased (50-78%) EstA-induced NO production. Similarly, EstA induced TNF-alpha, IL-1 beta and IL-6 mRNA expression in RAW 264.7 macrophages in a dose-dependent manner, and pre-treatment of the cell cultures with specific NF-kappaB, p38 and ERK 1/2 MAPK pathway inhibitors significantly decreased EstA-induced TNF-alpha, IL-1 beta and IL-6 protein production. Furthermore, immunoblot analysis revealed the degradation of the inhibitory kappa B (IKB-alpha) in response to EstA stimulation. Taken together, our data suggests that EstA protein is a novel inducer of NO and pro-inflammatory cytokines by activating the NF-kappaB, p38 and ERK 1/2 MAPK pathways during inflammatory responses. Future studies on the upstream protein kinases of the MAPK/NF-kappaB pathways and the kinetics of cytokine production will provide further details into the mechanism of EstA-induced inflammatory response.